Identification of stress-responsive transcription factors with protein-bound Escherichia coli genomic DNA libraries.
Identification of stress-responsive transcription factors with protein-bound Escherichia coli genomic DNA libraries.
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DOI：
10.1186/s13568-020-01133-0
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发表时间：
2020-11-02
期刊：
影响因子：
3.7
通讯作者：
Guo J
中科院分区：
文献类型：
--
作者：
Li X;Jiang X;Xu M;Fang Y;Wang Y;Sun G;Guo J
细菌启动子沿着操纵子是微生物响应环境胁迫变化而调控基因表达的关键元件。全基因组启动子DNA调控文库的开发是在需求的微生物报告方法，以监测任何给定的环境胁迫物质的存在。在这项研究中，我们利用大肠杆菌（E。coli）作为制备细胞裂解物和基因组DNA片段的模型系统。通过富集蛋白质结合的DNA片段构建荧光素酶报告基因库，我们发现在收集并测序的280个克隆中，有131个克隆含有启动子-35和-10保守序列和/或操纵子转录因子结合位点（TFBS）区域。为了证明所鉴定的克隆的功能性，已经证明了131个含有莱克萨结合序列的克隆中的5个响应于丝裂霉素C处理而被诱导。为了评估我们的文库作为功能筛选文库，培养80个随机挑选的克隆，并用和不用MMC处理，其中两个克隆显示具有大于两倍的诱导。此外，从90个克隆中鉴定出两个亚砷酸盐响应性克隆，一个具有众所周知的ArsR，另一个具有代谢诱导脂蛋白（OsmE 1）。新发现的osmE 1已被定量验证砷处理诱导的实时PCR的剂量响应和时间过程的方式。这种富集蛋白质结合的DNA荧光素酶报告文库和功能筛选有助于鉴定微生物中的应激响应转录因子。我们开发了包含E.大肠杆菌全基因组蛋白结合DNA作为增强子/操纵子调节下游荧光素酶响应压力。
Bacteria promoters along with operators are crucial elements in the control of gene expression in microbes in response to environmental stress changes. A genome-wide promoter DNA regulatory library is in demand to be developed for a microbe reporter method to monitor the existence of any given environmental stress substance. In this study, we utilized Escherichia coli (E. coli) as a model system for the preparation of both cell lysates and genomic DNA fragments. Through enriching protein-bound DNA fragments to construct luciferase reporter libraries, we found that, of 280 clones collected and sequenced, 131 clones contained either the promoter-35 and -10 conservative sequences and/or an operator transcription factor binding sites (TFBS) region. To demonstrate the functionality of the identified clones, five of 131 clones containing LexA binding sequence have been demonstrated to be induced in response to mitomycin C treatment. To evaluate our libraries as a functional screening library, 80 randomly picked up clones were cultured and treated with and without MMC, where two clones were shown to have greater than twofold induction. In addition, two arsenite-responsive clones were identified from 90 clones, one having the well-known ArsR and another having the osmotically inducible lipoprotein (OsmE1). The newly discovered osmE1 has been quantitatively validated to be induced by arsenite treatment with real-time PCR in a dose response and time course manner. This enriching protein-bound DNA luciferase reporter libraries and functional screening facilitate the identification of stress-responsive transcriptional factors in microbes. We developed functional libraries containing E. coli genomic-wide protein-bound DNA as enhancers/operators to regulate downstream luciferase in response to stress.
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DOI：
10.1136/bmj.k3310
发表时间：
2018-08-29
期刊：
BMJ (Clinical research ed.)
影响因子：
--
作者：
Chowdhury R;Ramond A;O'Keeffe LM;Shahzad S;Kunutsor SK;Muka T;Gregson J;Willeit P;Warnakula S;Khan H;Chowdhury S;Gobin R;Franco OH;Di Angelantonio E
通讯作者：
Di Angelantonio E
影响因子：
4
作者：
Inukai S;Kock KH;Bulyk ML
通讯作者：
Bulyk ML
DOI：
10.1016/j.bbapap.2005.06.006
发表时间：
2005-08-10
影响因子：
3.2
作者：
Kriukiene, E;Lubiene, J;Lubys, A
通讯作者：
Lubys, A
影响因子：
--
作者：
Brenowitz, M;Senear, D F;Kingston, R E
通讯作者：
Kingston, R E
ArsR 结合序列中非共有碱基对对 ArsR-DNA 结合和砷介导的转录诱导的贡献
影响因子：
5.6
作者：
Chen, Xingjuan;Jiang, Xin;Li, Xianqiang
通讯作者：
Li, Xianqiang